Shigella spp. cause disease by invading and multiplying within human colonic epithelial cells. Successful infection requires appropriate timing of virulence gene expression and the efficient acquisition of nutrients within the host; however, the sensing and acquisition of carbon sources and other nutrients by Shigella during infection is poorly understood. This application focuses on the role of carbon metabolism pathways in the virulence of Shigella flexneri. Our data suggest that S. flexneri uses information about the available carbon sources to determine the attachment and initial steps in invasion of host epithelial cells. Once inside the host cells, the bacteria must adjust their metabolism to take advantage of the different carbon sources available in the intracellular environment. The carbon and other nutrient sources present in the host cell cytoplasm and the pathways used by intracellular Shigella to obtain these sources are largely unknown. The first specific aim is to define the carbon and nutrient sources available to S. flexneri in the intracellular environment of the host. Using metabolomics, we can assess the nutrients present in the cytoplasm of uninfected cells and follow changes in the metabolome during the course of infection. Once we have identified the carbon sources that are present, our second aim is to determine the pathways the bacteria use to assimilate these nutrients. We will use proteomic and transcriptomic analysis of the intracellular bacteria to define the carbon metabolism genes expressed by intracellular S. flexneri. These will be complemented by genetic analysis to determine which of the expressed pathways are required for, or contribute to, intracellular growth. We will construct mutants that are defective in one or more of the carbon metabolism genes expressed intracellularly and test their ability to invade and cause plaques. The third aim is to determine the mechanism by which a regulator of central carbon metabolism, Cra, is linked to S. flexneri invasion and cell-to-cell spread. A mutation in cra markedly increased S. flexneri adherence to epithelial cells but limited cell-to-cell spread, pointing to a link between S. flexnei carbon metabolism and virulence. We will use genetic and biochemical characterization of the cra mutant to determine the mechanism of Cra's effect on virulence. Taken together, the data generated in this study will provide essential information on carbon metabolism and its role in Shigella virulence, and will contribute significantly to our broader understanding of the physiology and metabolism of S. flexneri in the host cell environment. Such data are applicable to the design of therapeutics targeting intracellular S. flexneri, as well as to the design of vaccines based on antigens expressed when the bacteria are growing within host cells.